This invention relates to a new .beta.-galactosidase enzyme which in active form is used to digest or hydrolyze lactose into glucose and galactose. The new .beta.-galactosidase is stable at acidic pH values predominating in the digestive organs of human beings and is stable at ambient and elevated temperatures on storage. This invention further relates to a fermentation process for the production of this new .beta.-galactosidase by cultivating a microorganism of the genus Penicillium and particularly Penicillium multicolor.
.beta.-Galactosidase is an enzyme which hydrolyzes the lactose present in the milk of mamalian animals to produce glucose and galactose. In recent years, this enzyme has called public attention as it is useful to improve or treat therapeutically the dyspeptic disorder of infants which are not capable of digesting lactose. The enzyme is also useful in the processing of various milk products.
Some kinds of .beta.-galactosidase are already produced by commercial processes comprising cultivating a bacterium, yeast or fungus and isolating these .beta.-galactosidases from the culture or culture broth of the microorganism. However, all of the known .beta.-galactosidases are not satisfactory because they have one or more certain drawbacks with respect to their activity and stability as well as the yield of production.
Known .beta.-galactosidases produced by microorganisms include the ones produced by bacteria such as lactic-acid bacteria and the ones produced by yeasts such as Saccharomyces fragilis. The known .beta.-galactosidases produced by bacteria and yeasts are intercellular enzymes which remain within the cell body of the bacterium or yeast, therefore it is necessary to remove and extract these enzymes out of the cell body of these microorganisms. The preparation of these intercellular enzyme products needs a number of production stages, and besides, it is difficult to recover these intercellular enzyme products in high yield.
On the other hand, it is known that some .beta.-galactosidases are produced as the extracellular enzyme by fungi of the genus Aspergillus such as Aspergillus niger (see Japanese patent publication No. 21078/74), Aspergillus oryzae (Japanese patent application pre-publication "Kokai" No. 151385/76), Aspergillus awamori (Japanese patent publication No. 22709/74), by fungi of the genus Macrophomina (Japanese patent application pre-publication "Kokai" No. 117677/76), by fungi of the genus Sclerotium (Japanese patent application pre-publication "Kokai" No. 116291/74) and by fungi of the genus Tricoderma (Japanese patent application pre-publication "Kokai" No. 62688/74).
Among these known .beta.-galactosidases which are produced as the extracellular enzyme, the ones which are produced by Aspergillus niger and Aspergillus awamori as well as by the fungi of the genus Sclerotium are acidic .beta.-galactosidase which show the optimum pH at a highly acidic value of 2 to 3.5 and hence are of limited applications.
Furthermore, the known .beta.-galactosidases produced by Aspergillus oryzae and by fungi of the genus Macrophomina or the genus Tricoderma are differentiated from the new .beta.-galactosidase which is produced by Penicillium multicolor according to this invention, with respect to their optimum temperature value, the optimum pH value, the temperature range and the pH range at which these enzymes are stable. All of the above-mentioned known fungi, except Aspergillus oryzae FERM-P 1680, have a very poor ability to produce the .beta.-galactosidase as the extracellular enzyme.
Aspergillus oryzae FERM-P 1680 has a favorably high ability to produce .beta.-galactosidase in a high potency, but the .beta.-galactosidase produced by this particular strain can be inactivated at acidic pH value of 2.5 to 3.0 and hence shows a lower stability at the acidic pH values, as compared to the new .beta.-galactosidase according to this invention. When the .beta.-galactosidase of Aspergillus oryzae FERM-P 1680 is administered as a digestive drug to men, it is often useless as it is very likely to be inactivated at a pH value of 2.5 to 3.0 which are normally predominating in the cavity of the stomach.
In general, enzymatic preparations containing known .beta.-galactosidase are of low stability upon storage. There are methods of stabilizing these enzymatic preparations by freezedrying (see Japanese patent publication No. 50390/72, for example) and methods of stabilizing these enzymatic preparations by incorporating inositol therein (see Japanese patent application pre-publication "Kokai" No. 9783/76) in order to prevent the degradation of the .beta.-galactosidase.
Moreover, it is also known that Penicillium frequentans Westling (identified as FERM-P 3085) produces a .beta.-galactosidase which is stable at a pH value of 3 to 5 at 4.degree. C., the optimum pH being 3.5 to 4.5; Penicillium luteum Sopp (identified as FERM-P 3091) produces a .beta.-galactosidase which is stable at a pH value of 3.5 to 8 at 4.degree. C., the optimum pH being 4.0 to 5.0; Penicillium citrinum ATCC 9849 (identified as FERM-P 3086) produces a .beta.-galactosidase which is stable at a pH value of 3.5 to 8 at 4.degree. C., the optimum pH being 4.0 to 5.0; Penicillium glaucum Link (identified as FERM-P 3090) produces a .beta.-galactosidase which is stable at a pH value of 3.5 to 8 at 4.degree. C., the optimum pH being 4.0 to 5.0; Penicillium chrysogenum Thom IFD 4626 (identified as FERM-P 3088) produces a .beta.-galactosidase which is stable at a pH value of 3 to 8 at 4.degree. C., the optimum pH being 4.0 to 5.0; and Penicillium notatum Westling (identified as FERM-P 3087) produces a .beta.-galactosidase which is stable at a pH value of 3.5 to 8 at 4.degree. C., the optimum pH being 4.0 to 5.0. However, these Penicillium species can only produce the above-mentioned .beta.-galactosidases in poor yield which is not commercially satisfactory. Additionally, the known .beta.-galactosidases produced by these Penicillium species are not entirely satisfactory because they are not stable at pH values of 2.5 to 3.0 predominating in the stomach of human beings and because they can be inactivated completely by heating at an elevated temperature of about 65.degree. C. for 15 minutes at pH 6.